Abstract

Introduction and Objective: A significant clinical challenge with LC-MS/MS is the potential for matrix effects that cause interferences or impact ionization efficiency. Stable isotope-labeled internal standards are frequently used to compensate for matrix effects and to increase the accuracy of quantitation. The use of a labeled internal standard that co-elutes with the drug being monitored can potentially offset patient specific matrix effects (co-eluting concomitant medication, etc.) that may occur at the retention time of the analyte of interest. Complications in the use of deuterium-labeled internal standards can arise from hydrogen-deuterium scrambling in the collision cell at the selected transitions or in the ion source. In this study, we examined deuterium labeled 25-Hydroxyvitamin D, testosterone, and other compounds of clinical significance by LC-MS/MS at multiple transitions. We investigated reproducibility of the scrambling ratio and influences on scrambling of different LC-MS systems (tandem quadrupole vs. quadrupole time-of-flight), matrix selection, concentration, and deuterium placement in the internal standard.

Serum Extraction:
200μL of sample in serum + 200μL of methanol, vortexed to mix.
Added 1mL of heptane, vortexed for 30sec,
Centrifuged for 4min at 3000rpm
900μL of top layer dried under nitrogen
Reconstituted in 100μL of ethanol

Comparisons of 25-Hydroxyvitamin D2 and D3 Deuterium Scrambling

25-Hydroxyvitamin D2 25-Hydroxyvitamin D2-d3 25-Hydroxyvitamin D2-d6

25-Hydroxyvitamin D3 25-Hydroxyvitamin D3-d6

Labeled 25-Hydroxyvitamin D2 and D3 Scrambling in Serum

Compound

Label

System

Concentration
μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/ dn

25-Hydroxyvitamin
D2

d3

Xevo G2

2

398→379

398→380

28.6

0.2

398→379

398→380

35.4

6410

5

416→397

416→398

2.8

416→379

416→380

19.7

398→379

398→380

30.4

50

416→397

416→398

2.8

416→379

416→380

20

398→379

398→380

30.5

d6

6410

5

419→400

419→401

2

419→382

419→383

8.8

401→382

401→383

5.9

50

419→400

419→401

2

419→382

419→383

9

401→382

401→383

5.4

25-Hydroxyvitamin
D3

d6

6410

2.5

407→388

407→389

4

407→370

407→371

18.8

389→370

389→371

9.2

Vitamin D in Serum on 6410

Vitamin D in EtOH Scrambling on Xevo G2

Investigation of Method, Instrument, and Concentration Effects on Scrambling for Vitamin D

Compound

Method

Instrument

Concentration
μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/ dn

d3 labeled 25-Hydroxyvitamin D2

Infusion

Q-Tof

10

398→379

398→380

29.7

5

30.9

10

27.1

LC

6410

100

30.4

33

30.2

Transitions Comparisons for Native and Labeled 25-Hydroxyvitamin D2 and D3 in EtOH on 6410

Parent →Water loss

Compound

Label

Concentration
μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/ dn

25-Hydroxyvitamin D2

d3

100

416→397

416→398

2.9

d6

100

419→400

419→401

2

native

50

413→394

413→395

0.5

25-Hydroxyvitamin D3

d6

50

407→388

407→389

4

native

100

401→382

401→383

0.5

Parent →2 Water losses

Compound

Label

Concentration
μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/ dn

25-Hydroxyvitamin D2

d3

100

416→379

416→380

19.5

d6

100

419→382

419→383

8.9

native

50

413→376

413→377

0.5

25-Hydroxyvitamin D3

d6

50

407→370

407→371

18.9

native

100

401→364

401→365

0.3

Water Loss →2 Water losses

Compound

Label

Concentration μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/ dn

25-Hydroxyvitamin D2

d3

100

398→379

398→380

30.4

d6

100

401→382

401→383

5.4

native

50

398→376

398→377

0.4

25-Hydroxyvitamin D3

d6

50

389→370

389→371

11.2

native

100

383→364

383→365

0.3

Notes: 25-Hydroxy D2-D6 water loss→2 water loss has same transition as 25-Hydroxyvitamin D3 parent→waterloss. Can be problem if compounds are not well resolved chromatographically

Note: Under optimized UPLC-Q-Tof conditions only water loss MS ions were detected. MS ion ratios changed for 25-Hydroxyvitam D when combined with mobile phase. Could detect ions without water loss when infusing.

It may be advisable to investigate at higher concentrations than normally analyzed to ensure that instrument sensitivity doesnot impact accuracy of scrambling determination.

Awareness of potential scrambling is important for proper internal standard selection. Scrambling may be mitigated or eliminated by altering instrument conditions and transition selection.

Deuterium-labeled internal standards are a viable option for LC-MS/MS analysis with selection of the appropriate transition. Deuterated standards can be more cost effective than 13C labeled internal standards, more widely available and with lower cost per test.13C labeled internal standards are most effective when deuterium scrambling issues can not be resolved.